Glycobiology is a newly emerging area of biotechnology. Most of the extracellular proteins of higher animals are glycoproteins, including proteins of pharmaceutical interest such as erythropoietin, tissue plasminogen activator, interleukins and interferons. The ubiquity and diversity of glycoproteins is matched by the breadth of functions that they have in a wide range of important biological processes. For instance, glycosylation plays an important role in hormone signal transduction and in the biological activity of immunoglobulins. Glycoproteins also play a structural role in connective tissues such as collagen. Glycosylation of proteins clearly represents one of the most important co- and post-translational events.
Glycoproteins are composed of a polypeptide chain covalently bound to one or more carbohydrate moieties. There are two broad categories of glycoproteins with carbohydrates coupled through either N-glycosidic or O-glycosidic linkages to their constituent protein. The N- and O-linked glycans are attached to polypeptides through asparagine-N-acetyl-D-glucosamine and serine (threonine)-N-acetyl-D-galactosamine linkages, respectively. Complex N-linked oligosaccharides do not contain terminal mannose residues. They contain only terminal N-acetylglucosamine, galactose, and/or sialic acid residues. Hybrid oligosaccharides contain terminal mannose residues as well as terminal N-acetylglucosamine, galactose, and/or sialic acid residues.
With N-linked glycoproteins, an oligosaccharide precursor is attached to the amino group of asparagine during peptide synthesis in the endoplasmic reticulum. The oligosaccharide moiety is then sequentially processed by a series of specific enzymes that delete and add sugar moieties. The processing occurs in the endoplasmic reticulum and continues with passage through the cis-, medial- and trans-Golgi apparatus (FIGS. 1A and B).
The regulation of the glycosylation process is complex because it contains both synthetic and degradative steps that are controlled by very specific enzymes. Currently, the regulation of glycoprotein synthesis and processing is not well understood.
Glycosylation in the Baculovirus Expression System
It has been estimated that the baculovirus-polyhedrin protein can constitute up to 50% of the total protein mass at cell death. The polyhedrin gene is one of the most highly expressed viral genes described. One of the reasons for this high expression level is that the polyhedrin gene is under the transcriptional control of a very strong promoter. Replacement of the polyhedrin gene open-reading-frame (ORF) with the ORF of a foreign gene under the control of the polyhedrin gene promoter results in high levels of expression of the foreign gene product. Production levels as high as 1 mg/10.sup.6 cells have been obtained. This method of producing foreign proteins is referred to as the baculovirus expression vector system (BEVS).
Hundreds of proteins have been expressed in stationary insect cell cultures with the baculovirus expression vector system (BEVS). There is substantial pharmaceutical interest in using the BEVS to produce commercial products in insect cells. The BEVS has several advantages as a recombinant protein production system, such as 4-6 weeks from gene isolation to BEVS expression, high production levels and the absence of adventitious viruses (commonly found in mammalian tissue culture cells). Equally important is the fact that insect cells are able to recognize the co- and post-translational signals of higher eukaryotes, resulting in processing such as phosphorylation, proteolytic processing, carboxyl methylation, and glycosylation. Of all these co- and post-translational processing events, glycosylation has been found to have the greatest influence on many of the physical and functional properties of proteins.
Altering the type of glycan modifying a glycoprotein can have dramatic affects on a protein's antigenicity, structural folding, solubility, and in vivo bioactivity and stability. Also, varying the number and composition of the oligosaccharide moieties can significantly alter the physical characteristics for many glycoproteins. In particular, it has been demonstrated that terminal sialic acid residues play an extremely important role in defining the in vivo biological activity of many glycoproteins. For example, terminal sialic acid residues have been demonstrated to be very important in defining the immunogenicity of glycoproteins.
The absence of sialic acid has been found to influence the biological activity of many proteins. In particular, the specific activities of proteins, such as tissue plasminogen activator (used clinically to dissolve blood clots) and erythropoietin (which stimulates maturation of red blood cells), have been found to be dramatically altered by the removal of terminal sialic acid residues. Furthermore, the specific recognition of oligosaccharide moieties is the primary mechanism for protein clearance from the circulatory system. Therefore, differences in the oligosaccharide structure, particularly the presence or absence of sialic acid, can significantly affect both the in vivo and in vitro properties of glycoproteins. Thus, if insect cells are used to produce therapeutic glycoproteins, it is critical to generate glycoproteins with terminal sialic acid residues.
Experience with the expression of N-linked glycoproteins using the BEVS clearly indicates that insect cells generally recognize the same signals for glycosylation sites as mammalian cells. The N-linked glycosylation pathway is outlined in FIGS. 1A and B. Glycosylation begins with the attachment of the dolichol-phosphate precursor oligosaccharide. Following this initial step, there is efficient removal of glucose residues by .alpha. glucosidase I and II and subsequent removal of mannose residues with endoplasmic reticulum mannosidase and Golgi mannosidase I. This glycan trimming process appears to progress in a proficient fashion in lepidopteran larvae and tissue culture cells.
Following these trimming events, mammalian glycan processing is typically subject to the sequential enzymatic addition of N-acetylglucosamine (GlcNAc), sometimes fucose, followed by galactose (Gal) and sialic acid residues (FIGS. 1A and B). However, mammalian glycoproteins that normally have complex glycans with terminal sialic acid residues when they are produced in mammalian cells, are expressed in the BEVS in insect cells with oligosaccharides containing high mannose (Man.sub.8-5 GlcNAc.sub.2) or paucimannose (Man.sub.2-3 GlIcNAc.sub.2) structures (note the absence of sialic acid residues). Some of the structures contain .alpha.1,6 linked fucose and/or terminal GlcNAc residues.
Early studies of N-linked glycoproteins expressed in the BEVS suggested that insect cells were not able to add GlcNAc, Gal or sialic acid residues (Wathen et al., 1989; Kuroda et al., 1990; Kretzschmar et al., 1994). However, the enzymes required for the addition of GlcNAc and Gal residues have been identified in insect cell lines derived from B. mori (Bm-N), Mamestra brassicae (IZD-Mb-05030 referred to as Mb) and Spodoptera frugiperda (IPLB-SF21AE referred to as Sf-9 and Sf-21). .beta.1,2-N-acetylglucosaminyltransferase I (GlcNAc-T-I) activity has been found in Bm-N, Mb, Sf-9 and Sf-21 tissue culture cells (Altmann et al., 1993). However, it should be noted that the high level of GlcNAc-T-I activity found in the Bm-N, Mb and Sf-21 tissue culture cells by Altmann et al. (1993) was not reflected in the N-linked oligosaccharide structures associated with cell membranes characterized by Kubelka et al. (1994). Only a small percentage of the membrane-associated structures had terminal GlcNAc residues.
Following the addition of the first GlcNAc residue, typically two additional terminal mannose residues are removed by the action of a Golgi mannosidase II (FIG. 1 B). Altmann and Marz (1995) demonstrated .alpha.-D-mannosidase II activity in Bm-N, Sf-21 and Mb insect cell lines. The enzymatic activity appeared to be membrane-bound. Ren et al. (1997) purified this enzyme from Sf-21 cells and found similar properties to those reported by Altmann and Marz (1995).
The resulting glycans with terminal GlcNAc residues can be fucosylated. Staudacher et al. (1992) found fucosyltransferase activity in Mb cells that transferred fucose to the innermost GlcNAc residue with .alpha.1,6 and .alpha.1,3 linkages. In addition, they identified fucosyltransferase activity for .alpha.1,6 fucosyl linkages in extracts from Bm-N and Sf-9 cells.
Altmann et al. (1993) also investigated the fucosyltransferase activity in Mb tissue culture cells. Based on substrate preference, they concluded that the go-signal for the lepidopteran fucosyltransferase was a GlcNAc residue on the .alpha.1,3 arm, the product of the GlcNAc-T-1 activity that they had previously found in these cells. Despite this apparent requirement by fucosyltransferase, Kubelka et al. (1994) found a low percentage of structures with GlcNAc residues on the .alpha.1,3 arm but a high percentage of fucosylated structures.
An explanation for this apparent contradiction is that following addition of GlcNAc to the .alpha.1,3 arm and subsequent fucosylation, the terminal GlcNAc residue is removed by .beta.-N-acetylglucosaminidase (GlcNAcase). In contrast, Ogonah et al. (1996) found an abundance of glycan structures with terminal GlcNAc residues attached to human interferon-.gamma. produced in Estigmene acrea (Ea-4) but not in Sf-9 tissue culture cells. The reason for this is that Estigmene acrea cells contain little or no GlcNAcase activity.
The digestion with a GlcNAcase is consistent with the paucimannose structures attached to secreted alkaline phosphatase during synthesis in five insect larvae and five cell lines (Kulakosky et al., 1998b). It was observed that all larval and cell culture samples except the Sf-21 cell culture samples contained high concentrations of fucosylated and nonfucosylated paucimannose structures that lacked terminal .alpha.1,3 mannose. This suggested that, following the removal of the GlcNAc from the .alpha.1,3 arm, an .alpha.1,3 mannosidase might remove the terminal mannose, leaving a structure that could not be further modified.
However, in the absence of GlcNAcase, an additional GlcNAc residue can be added to the .alpha.1,6 arm through the action of .beta.1,2-N-acetylglucosaminyltransferase II (GlcNAc-T-II). Altmann et al. (1993) reported finding low levels of GlcNAc-T-II activity in Bm-N, Mb, Sf-9 and Sf-21 tissue culture cells. Their data indicated that the GlcNAc-T-II was responsible for the addition of a GlcNAc residue .beta.1,2 linked to the .alpha.1,6 arm.
The resulting structures would be substrates for the enzymatic addition of Gal residues. A .beta.1,4 galactosyltransferase has been reported in BTI-Tn-5b 1-4 (High Five.sup.a), Sf-9 and Mb tissue culture cells. This suggests that insect cells have the necessary enzymatic machinery for processing complex glycans containing terminal Gal residues. However, very few recombinant glycoproteins produced in insect cells have been found to have oligosaccharides with terminal Gal residues.
There is considerable interest in producing N-linked glycoproteins that have glycan structures terminating with sialic acid residues. However, the requisite sialyltransferase activity has not been reported in insect cells. This fact and the typical lack of sialylated glycans with BEVS-expressed N-linked glycoproteins have raised questions concerning the presence and/or concentration of sialyltransferase in insect cells.
Several strategies have been used to extend the processing of glycans in insect cells to achieve glycans containing additional GlcNAc, Gal and sialic acid residues. One approach has been to co-infect cells with a recombinant baculovirus expressing a glycosyltransferase and one expressing an N-linked glycoprotein. For instance, Wagner et al. (1996b) co-infected Sf-9 cells with a baculovirus expressing a human GlcNAc T-I and one expressing fowl plaque virus hemagglutinin. The co-expression of the GlcNAc T-1 resulted in a four-fold increase in glycans with terminal GlcNAc residues attached to the hemagglutinin.
Using a bovine Gal T-1 enzyme expressing BEVS, Jarvis and Finn (1996) detected terminal Gal residues on glycans attached to gp64, an AcMNPV structural glycoprotein. In the absence of Gal T-1 expression, lectin-binding assays detected terminal mannose and GlcNac residues but no Gal residues on the gp64 glycans. Similar results were obtained during wild-type AcMNPV replication in transformed (also referred to as stably transfected) Sf-9 cells expressing an integrated Gal T-1 gene.
Jarvis and co-workers have also used a combination of transformed Sf-9 cells and a baculovirus expressing mammalian genes involved in glycan processing. Jarvis and Finn (1996) constructed a BEVS expressing GlcNAc T-1, and then used this virus to infect cells previously transformed with Gal T-1. The resulting addition of GlcNAc and Gal residues to the gp64 glycans indicated that the substrates UDP-GlcNAc and UDP-Gal might not be limiting in Golgi of Sf-9 cells.
Jarvis, Kawar and Hollister (1998) constructed a BEVS expressing a mammalian .alpha.2,6 sialyltransferase gene. They used this virus to infect transformed Sf-9 cells expressing Gal T-1. The resultant glycans attached to the baculovirus gp64 protein contained Gal and terminal sialic acid residues as determined by lectin-binding analyses. The results suggest that the expression of foreign sialyltransferase can be used to produce recombinant N-linked glycoproteins with terminal sialic acid residues. In addition, the results suggest that Sf-9 cells contain the CMP-sialic acid substrate and that it is transported from the nucleus to the Golgi apparatus. However, the lectin analyses employed are questionable and have not allowed quantitative measurements.
In the absence of genetic engineering, the question remains unanswered as to whether lepidopteran insect cells have the metabolic potential for mammalian-like complex glycosylation with terminal sialic acid residues. Clearly, GlcNAcase and .alpha.1,3 mannosidase activities could be used to explain the abundance of paucimannose structures detected in many BEVS studies with glycoproteins. However, it occurred to the inventor that processing of glycans might be significantly influenced by cell type, cell culture media components, culture conditions, and the properties of the baculovirus as well as the properties of the protein being expressed. In addition, one needs to keep in mind that during BEVS production of recombinant proteins, the cell is undergoing apoptosis. Considerable research in this area is required to reach a basic understanding of the underlying factors that control the glycan processing with individual glycoproteins.
A part of this understanding will come from the study of the rare BEVS expressed recombinant glycoproteins that process glycans beyond paucimannose structures. For instance, with IgG expressed in BTI-TN-5B1-4 cells, approximately 20% of the glycans have one terminal Gal residue and 65% of the glycans have one or more terminal GlcNAc residues.
The first publication in which sialylation occurred with the BEVS in cell culture was by Davidson et al. (1990). They characterized glycans attached to human plasminogen (HPg) during BEVS expression in Sf-21 cells. Using a combination of lectin-blotting, anion-exchange liquid chromatography and glycosidase digestions, they found that approximately 40% of the glycans attached to HPg contained terminal sialic acid residues. Davidson and Castellino (1991) expressed this same protein in the Mamestra brassicae cell line, IZD-MB0503, and found that 53% of the glycans attached to HPg contained terminal sialic acid residues. They also detected HPg sialyated glycans produced in the CM-1 line derived from Manduca sexta.
Sridhar et al. (1993) reported that expression using an earlier and weaker viral gene promoter (the MP promoter instead of the polyhedrin promoter) produced the .beta. subunit of human chorionic gonadotropin with some sialylation. However, the level of sialylation was less than observed in mammalian cells. Vandenbroeck et al. (1994), using lectin-blotting analyses, detected terminal sialic acid residues attached to the glycans of BEVS expressed porcine interferon-.gamma. produced in Sf-9 cells. Russo et al. (1998), also using lectin-blotting analyses, reported terminal sialic acid residues on glycans attached to a bovine leukemia virus envelope glycoprotein produced in Sf-21 cells.
Ogonah et al. (1996) reported complex glycosylation of human interferon-.gamma. produced with the BEVS. The complex glycosylation included the synthesis of terminal N-acetylyglucosamine and galactose but not sialic acid. Complex processing was obtained in E. acrea but not S. frugiperda (Sf-9) tissue culture cells.
A problem with lectin-blotting analyses, the analytical tool used for the majority of the glycan analysis described above, is that it does not allow for quantitative determinations. In addition, although appropriate controls were included in all lectin-blotting experiments, glycobiologists advise that false-positive data are not uncommon with lectin-blotting studies. The potentially suspect results obtained from lectin-blotting experiments must be confirmed with results from other techniques to be convincing.
Although there have been very few investigations concerning endogenous processing (without BEVS) of glycoproteins in lepidopteran cells, there is a report by Kato et al. (1994) in which they found evidence of glycans with terminal sialic acid residues. They reported a 130 k glycoprotein from the hemolymph of B. mori larvae that was present in "active" and "inactive" haemagglutination forms at different stages of larval development. Using chemical and enzymatic removal of sialic acid residues from this purified lectin and HPLC to quantitate sialic acid contents, they found high levels of sialic acid residues on glycans of the inactive form and no sialic acid residues on the active form. Similarly, sialylated glycans that are cell type-specific and developmentally regulated have also been identified in Drosophila melanogaster (Roth, 1992) using lectin-gold histochemistry and gas liquid chromatography-mass spectroscopy.
Based on the above-mentioned studies, it appears that insects have the potential to process N-linked mammalian glycoproteins, with glycan structures similar to those attached during production in mammalian cells. However, no one has provided a reliable expression system that can realize this potential.
Baculovirus Expression Vector System (BEVS) and Microgravity/Low Shear Bioreactors
There has been a great deal of interest in the production of recombinant glycoproteins with the various expression vector systems. The baculovirus expression vector system (BEVS) employs lepidopteran larvae and their derived cell culture (referred to herein simply as insect cells). Because insect cells have been shown to recognize the signal sequences and possess the metabolic pathways for processing glycoproteins in a manner similar to mammalian cells, there has been a great deal of interest in using the baculovirus expression vector system to produce recombinant N-linked glycoproteins.
Insect tissue culture cells have difficulty growing in "traditional" bioreactors. The major limitation to the scale up of insect cells has been providing sufficient oxygen without damaging the cells. Insect cells in culture have a 3-10 fold higher oxygen demand than mammalian cell cultures. Upon infection of those cells with recombinant virus, the demand for oxygen can be increased 50%-100%. For small-scale spinner flask cultures, the surface area-to-volume ratio is large enough that diffusion alone can supply sufficient oxygen. However, as the volume of a bioreactor increases, the surface area-to-volume ratio decreases, leading to oxygen limitation. This limitation leads to decreases in cell density and lower yields in production.
In order to overcome the large demand for oxygen, aeration by bubbling air (oxygen or a mixture) through the culture medium has been used. However, this method can significantly damage insect cells due to turbulence. Insect cells are much more shear-sensitive than microbial cells due to their larger size and lack of a cell wall. Virus-infected insect cells are even more shear-sensitive, since they swell to twice their original size upon virus infection. To overcome this dual problem of providing sufficient oxygen without damaging the cells, protective agents have been added to the medium. However, the problem has only been partially overcome.
As a result of these shear forces, the protein production levels observed in stationary cell cultures are not always obtained with suspension cell cultures. Until recently, the technology has not been available to evaluate these interactions. However, the development of the microgravity/low shear bioreactor, HARV, has made it possible to directly evaluate the effects of microgravity/low shear upon cellular function and structure.
Microgravity and Shear Forces
In vitro investigations have shown that environmental factors can influence oligossacharide processing. Goochee and Monica (1990) have reviewed cell culture studies in which environmental factors affected N-linked glycosylation. They discussed several alterations to glycosylation that were chemically induced, i.e. glucose starvation, hormones, and acidotropic amines. Further confirmation of the importance of slight changes in culture conditions came with the discovery of changes in glycoforms during batch culture and from batch to batch (Hooker et al., 1995).
Microgravity/low shear effects on glycosylation have not been studied. However, there have been several microgravity/low shear investigations in other contexts. For instance, Hymer et al. (1996) suggested that disparate post-translational modifications occurred in the rat PRL hormone following space flight. Bechler et al. (1992) and Fuchs and Medvedev (1993) observed increases in the production and secretion of interferon (a pharmaceutically important glycoprotein) by lymphocytes during space flight.
Microgravity can have an effect on structural organization of the endoplasmic reticulum and Golgi apparatus. For instance, Moore et al. (1987) found that corn cell endoplasmic reticulum development under microgravity was abnormal, with structures clumping into spherical and ellipsoidal masses. Similar endoplasmic reticulum disruptions were observed by Hilaire et al. (1995) with sweet clover cells cultured in a rotating clinostat. Under simulated microgravity, rice cell walls grew irregularly, and microgravity-mediated structural changes in chloroplast grana and mitochondria cristae have been noted. Similarly, experiments with Chlamydomonas reinhardtii illustrated microgravity-mediated changes in shape, structure and distribution of cell organelles.
It is known that hydrodynamic shear forces, which result when culturing conditions are scaled up in large bioreactors, influence the cytoskeletal structure of cultured cells. In 1988, Schurch et al. determined that shear forces generated in cell culture affect both cell shape and membrane integrity. Hydrodynamic shear induction of gene transcription and enzyme activity is well established. In addition, shear forces have been found to affect both the level of protein synthesis and the extent of glycosylation. The precise mechanism(s) responsible for these changes are unknown.
Recently, experiments were performed in which insect cells (Sf9) were cultured and infected with a recombinant virus expressing .beta.-galactosidase in the HARV bioreactor designed to simulate a microgravity/low shear environment. In this environment, the insect cells produced approximately 7-fold more .beta.-galactosidase protein than Sf9 cells cultured in shaker flasks. In addition, the Sf9 cells underwent substantial morphological and physiological changes with a sustained stationary phase.
Since the development of the BEVS in the early '80s, the BEVS has been shown to have a high potential for the commercial production of recombinant proteins. Hundreds of recombinant proteins have been expressed with the BEVS because of the high production levels, ease of purification and the recognition of higher eukaryotic co- and post-translational signal sequences by insect cells. Although insect cells possess N-linked glycosylation processing machinery, under most conditions complex glycosylation has not been obtained with the BEVS. A technology leading to increased efficiency of complex oligosaccharide processing of glycoproteins is needed to further the development of this viral expression system.